Treatment of cerebral metastases of HER2-positive (HER2+) breast cancer remains an unmet need. The development of novel HER2 targeting agents has revolutionized the treatment of HER2+ systemic disease; however, the efficacy of these targeted drugs is very limited in the brain microenvironment. Treatment is palliative in the majority of the cases, with survival rates varying between 4-12 months. This poor prognosis emphasizes the urgent need to unravel the mechanisms that underlie resistance of brain metastases (BM) to HER2 targeted drugs, in order to optimize therapeutic approaches in this setting. Recent preclinical studies indicate that ErbB3 (HER3) has a central role in promoting resistance to HER2 targeted therapies. We have discovered that HER3 and its activating ligand neuregulin-1 (NRG-1) are highly expressed in HER2+ breast cancer BM. Moreover, we have found that HER3 blockade enhances the efficacy of anti-HER2 therapy in the brain, resulting in significant tumor growth delay and improved survival. Building on these exciting preliminary findings, we now propose to unravel the mechanisms involved in HER3-mediated resistance to HER2 inhibition in the brain using well-characterized primary and patient-derived human HER2+ breast cancer cell lines, transgenic mouse models, and state-of-the-art imaging techniques. In Aim 1, we will examine the molecular mechanisms of NRG-1-dependent HER3 activation in the brain microenvironment. In Aim 2, we will investigate how the NRG-HER3 axis mediates resistance to anti-HER2 therapies. Lastly, in Aim 3 we will determine the effects of combinatorial NRG-1/HER3 and HER2 pathway inhibition in translational studies using brain metastasis models. To realize these aims, we have developed clinically relevant animal models of breast cancer BM, and powerful, non-invasive, high resolution imaging technologies that provide unprecedented molecular, cellular, structural and functional insights, and reveal various steps of BM progression. We will use these techniques and the unique collective expertise of our multidisciplinary team to uncover the role of the NRG-1/HER3 axis in mediating resistance to HER2 targeted therapies. Furthermore, we will validate the therapeutic benefit of combinatorial treatment strategies, which will directly inform clinical trials in patients with HER2+ breast cancr brain metastases, and will meet the urgent need for effective therapies.